Primary immune deficiencies with impaired cell-mediated cytotoxicity most commonly manifest as Familial Hemophagocytic Lymphohysticytosis (FHL), a life threatening disorder. FHL subjects harboring germline mutations in the genes encoding Syntaxin 11 (STX11, FHL-4) and Syntaxin Binding Protein 2 (STXBP2, FHL- 5) display abnormal Cytotoxic T-lymphocyte and Natural Killer cell activity owing to impaired lytic granule (LG) exocytosis. This process involves a highly orchestrated series of protein-protein interactions that culminate in fusion of the LG membrane with the plasma membrane, releasing perforin and other LG contents. STX11 and STXBP2 physically interact in as yet poorly characterized ways to orchestrate this fusion event. Due to the complexity of the cellular environment, it has been challenging to define the cellular and molecular defects underlying immunodeficiency in patients with STX11 or STXBP2 mutations. Toward this end, this proposal aims to establish the basic mechanisms through which STX11 and STXBP2 cooperate to effect LG fusion, and to use novel in vitro and ex-vivo fusion assays to understand how these molecular mechanisms are affected by FHL-associated mutations. To address these questions, we will carry out three Specific Aims. First, we will ask whether and how FHL-mutations in STX11 and STXBP2 affect physical interactions with one another and with other SNAREs. We will use a biochemical approach, performing pull-down, liposome co-flotation and surface plasmon resonance experiments to evaluate how mutations affect the structural requirements for protein- protein interactions. To assess the biological significance of our in vitro results, w will perform immunoprecipitation experiments using ex-vivo cells. Second, we will test the effect of FHL-mutations on intracellular membrane trafficking and lytic granule release. We will precisely localize STX11, STXBP2 and other key trafficking protein machinery under resting and activated conditions in ex-vivo cells from normal and FHL subjects using super-resolution STED microscopy. Additionally, we will set up a TIRF-based dual-color secretion assay to accurately evaluate at the single cell level the effects of FHL-mutations on LG release. This assay will also be used to explore novel strategies to rescue the impaired LG release in FHL cells. Third, we will develop novel methods to study the functional effect of FHL-associated mutations on LG secretion with high resolution and without the complexity of the cellular environment. Toward this end, we will perform liposome-liposome fusion assays, specifically designed to assess the molecular function of Stx11 and STXBP2 proteins in LG granule secretion. This unique simplified system, will serve to elucidate the mechanism by which FHL mutations affect membrane fusion. In summary, this project will combine basic biochemistry using recombinant proteins and in-vitro fusion assays with cell-biological experiments in patient-derived cells to provide new insights into the functional consequences of the patients' genetic defects. Results from these studies will also provide insights that can be applied to diagnosis and targeted therapies for FHL.